Single-strand conformation polymorphism (SSCP) is the most widely used method for mutation scanning. With SSCP, single-base sequence changes can be detected by altered electrophoretic migration of one or both single strands on a non-denaturing gel. SSCP does not detect all sequence changes with one electrophoresis condition and its sensitivity is a complex function of sequence context and size (Glavac and Dean, 1993; Hayashi and Yandell, 1993; Hongyo et al., 1993; Liu and Sonuner, 1994; Michaud et al., 1992; Sarkar et al., 1992a; Sarkar et al. 1992b; Sheffield et al., 1993; Takahashi-Fujii et al., 1993).
Previous work suggested that the idiosyncratic nature of SSCP sensitivity is a function of both the distribution of mobility of single-base changes and the mobility of the wild type sequences relative to that of all single-base changes (see FIG. 1). For a 200 bp segment, there are 600 possible variants that differ by a single-base substitution. If it were possible to generate all 600 possible variants and to plot the mobility in units of band widths, it is apparent that the sensitivity of SSCP will be less for a segment in which the mobility of the wild type sequence is close to the mode (see FIG. 1A). If the variance of mobility is wider (FIG. 1B), SSCP sensitivity, on average, will be higher than with the first condition. However, this is not necessarily so, because the location of the wild type sequence within the distribution is also critical.
At least two ways to increase the sensitivity of SSCP have been described. In one approach, SSCP is hybridized with another method in order to generate the redundancy of mutation-containing segments necessary to detect virtually all mutations. For example, in dideoxy fingerprinting (ddF), SSCP is combined with Sanger dideoxy sequencing (Lin and Sommer, 1994; Sarkar et al., 1992b). SSCP can also be combined with restriction endonuclease fingerprinting (REF) (Liu and Sommer, 1995) and with bi-directional dideoxy fingerprinting (bi-ddF) (Liu et al., 1996). A Sanger dideoxy termination reaction is performed with one dideoxy terminator. The terminated single-stranded segments are electrophoresed through a non-denaturing gel. The ladder of segments subsequent to the mutation contain the same mutation with different 3' ends. In a second approach, SSCP is performed under two or more conditions. Typically, two temperatures are utilized, and occasionally, two temperatures with and without glycerol (Glavac and Dean, 1993; Liu and Sommer, 1994).
Recently, we reported that the pattern of SSCP shifts varied markedly when HEPES was added to standard TBE buffer (Liu and Sommer, 1998). The correlation coefficient (R) between these two conditions was 0.46. These results hint that sugar/base and sugar/sugar interactions are more important than secondary structure, which should not be much affected by the addition of HEPES.
Herein, we report a detailed analysis of gel matrix, running buffer, temperature, and additive to search for a set of sensitive and complementary electrophoretic conditions for SSCP analysis. ddF was utilized in order to provide a very large sample of mutation-containing segments for analysis. From the data, five conditions were chosen and SSCP.sub.5 analysis was performed with 100% sensitivity in two blinded analyses.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.